Systems and methods of quantitative phase microscopy (QPM) are known that employ digital holography for studying the internal structures of biological cells without requiring the use of exogenous staining. Systems and methods of diffraction phase microscopy (DPM) based on off-axis point diffraction interferometry that allow quantitative phase imaging through a combination of single-shot imaging and a near-common-path geometry are also known.
In such known systems and methods of DPM, a technique can be employed in which an off-axis hologram image is recorded between two optical waves that are both derived from light transmitted through a sample. The two optical waves are the zero-th and first order beams that are generated when the transmitted light is incident upon an optical grating, which is the point where the optical wave paths diverge. The optical waves recombine at a detection plane after the zero-th order beam has passed through a Fourier-plane pinhole spatial filter, allowing the zero-th order beam to perform the function of a plane reference beam in the imaging plane. This technique has been characterized as having sub-nanometer path-length stability and millisecond-scale acquisition time.
Some known QPM systems employ an in-line point-diffraction-interferometry configuration that provides improved measurement resolution. In such a configuration, phase stepping (which is generally required for a non-off-axis geometry) is achieved by translating a transmission grating in a direction orthogonal to the optical axis. A variation of such a configuration that employs a pair of cube beam splitters (instead of transmission gratings), and several polarization-transforming optical elements, can simultaneously acquire two sample hologram images, phase-shifted with respect to each other, on different regions of a CCD detector.
In addition, systems and methods of QPM are known that employ a Zernike phase contrast approach, as opposed to holographic interferometry, to provide increased lateral resolution and reduced coherent noise. In such an approach, an illumination pattern consists of a series of discrete points evenly spaced around a circle, allowing phase stepping to be achieved by rotating a phase plate having structures etched onto a similarly sized circle. Such structures can help to mitigate halo and shade-off artifacts.
DPM is known to employ a Zernike phase contrast approach. This technique is referred to herein as instantaneous spatial light interference microscopy (iSLIM) in which a reference beam is passed through an annular aperture, and an optical grating and an amplitude mask are used to introduce a phase ramp between un-diffracted and diffracted components of an object wave, allowing an off-axis interferogram to be recorded between the object wave components. In this way, the stability of DPM can be combined with white-light illumination, which can diminish coherent speckle effects and enable spectroscopic imaging.
One drawback of the known methods of QPM/DPM that require a reference beam to be passed through a Fourier-plane pinhole spatial filter or an annular aperture as in iSLIM, is that they can create an alignment constraint that can increase the complexity of the systems, which is particularly problematic for non-specialists in the fields of QPM/DPM. Further, the size of the pinhole in the Fourier-plane pinhole spatial filter, or the size of the annular aperture, must typically be optimized with respect to the system optics, further contributing to the complexity of the systems and methods.
Some known systems and methods are known employ a self-referenced QPM technique, in which a transmitted sample wave is propagated through a Michelson interferometer that performs an image inversion after a double pass using an additional objective lens in one of its arms. In one known implementation of the self-referenced QPM technique, a phase-shifting procedure can be used to recover a sample-wave phase profile, while scanning a minor in the object arm to achieve an extended depth-of-field. To reduce temporal phase noise introduced between successive phase-stepped images, another known implementation of the self-referenced QPM technique is provided that uses Hilbert phase microscopy to determine the sample-wave phase profile. However, both known implementations of the self-referenced QPM technique described above employ separate sample and reference arms, making them susceptible to spatial phase noise.
Systems and methods of QPM can be used to provide the anisotropy of a sample under observation. The drawbacks of the known self-referenced QPM systems above include i) a system configuration that is not common-path, which can lead to spatial phase instability, and ii) requiring four serial measurements, which may be time consuming, requires sample to be stationary, and can introduce temporal phase noise.
It would therefore be desirable to have improved systems and methods of self-referenced quantitative phase microscopy (SrQPM) that avoid at least some of the drawbacks of the known systems and methods of QPM/DPM described above.